Need help for restriction enzymes

Hi
I got the problems about digest my insert and vector by restriction enzymes. I use BamHI and SalI to digest my vector (pET duet1) and insert genes (double digestion). Then Incubate at 37C for 2 hrs. Is that enough for 2 hrs?. After that I did ligation. Transformation and I got some colonies. Next step I did PCR colony screening but I did not find my expected band on the gel. I think it was because of restriction enzymes did not work properly. So any idea that I can confirm my restriction enzymes work? Please help.

Can you recover plasmid DNA from some of your transformants and digest it, looking for an insert?

Perhaps your PCR colony screening failed -- did you have a postive control?

Thank you for your advice Yes I did purify my digestion before ligation. I got colonies and I checked PCR colony screening and I got band but it was not the right size about 900 bps. I found band which lower than my expected size. It was false positive one I supposed to say. I don't know what should I do next. Any idea for me please

If your insert does not have an EcoRI or BspMI site, you can do the ligation (but I'd suggest you do 2 hr @ RT, not 37) then digest with the enzyme; this will linearise any single-cut plasmids, but not affect your double-cut, insert ligated plasmids.

To check the REs, just do single digestions and run on a gel. To test the ligase is working and the insert is correctly cut , treat some DNA ladder and some insert and run on agarose - the ladder should shift up and the insert should have at least a trimer-sized band.

When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.

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When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.

I completely agree. I used to just pick some small amount of a colony into 30 ul of PCR master mix, but then switched to picking about the same amount of colony into 50 ul of sterile water and using 1 ul of this suspension as template. My success rate went up dramatically.

When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.

I completely agree. I used to just pick some small amount of a colony into 30 ul of PCR master mix, but then switched to picking about the same amount of colony into 50 ul of sterile water and using 1 ul of this suspension as template. My success rate went up dramatically.